The present invention relates to an immunochemical method which permits simple semi-quantitative assay without diluting a sample containing an analyte, and an apparatus therefor.
As a method for the qualitative or quantitative determination of a trace amount of a substance contained in an in vivo sample such as blood or urine, immunochemical assay methods are generally used due to their high sensitivity. Of these methods, a so-called immunochromatographic method using chromatography is widely used in many fields, e.g., for clinical examination in a hospital and an assay test in a laboratory.
As a method of detecting an analyte by an immunochromatographic method, a variously labeled specific binding substance (antibody) is reacted with an analyte to be detected (antigen) on a chromatographic material to form a complex of the analyte and the labeled specific binding substance (antigen-antibody complex), and this complex is found (detected) by a variety of means. The label includes radioisotope, chromophore, fluorophore, enzyme, etc. The detecting means include a radiation detector, a spectrophoto meter etc. and visual detection.
JP-A-64-32169 discloses a qualitative assay method by immunochromatography using a colloidal-particles-labeled specific binding substance (antibody) which is chromatographically mobile and capable of generating visually detectable signal, and an apparatus therefor. This publication discloses one means which allows visual detection.
The method described in the above publication is a method for assaying the presence or absence of an analyte (antigen) in a sample or its amount, and comprises (a) bringing a sample containing an analyte (antigen) into contact with a chromatographic medium, (b) moving the above colloidal-particles-labeled substance (labeled antibody) on the chromatographic medium, to allow at least part of the colloidal-particle-labeled substance (labeled antibody) to move to a reactive site and cause a binding reaction, and (c) determining detectable response caused by the colloidal substance in the reaction site for denoting the presence or absence of the analyte (antigen) in the sample and its amount.
Japanese PCT Laid-open Publication No. 1-503174 discloses an apparatus provided with a labeled first antibody which is to specifically bind to an analyte to be detected (antigen) (to be referred to as xe2x80x9clabeled first antibodyxe2x80x9d hereinafter), and is freely mobile on a chromatographic medium in a wet state, and an unlabeled second antibody which is to specifically bind to the analyte (antigen) (the second antibody having an antigen binding portion different from that of the first antibody) (to be referred to as xe2x80x9cunlabeled second antibodyxe2x80x9d hereinafter), and fixed on the chromatographic medium, the apparatus being capable of detecting the presence of the analyte, in a detection area on the chromatographic medium by adding a liquid sample containing the analyte to one end of the chromatographic medium so that the liquid sample moves through the chromatographic medium, reacts with the labeled first antibody and then reacts with the unlabeled second antibody. The principle used in the above apparatus is a so-called (immunochemical) sandwich method.
For determining the amount of an analyte in a sample, conventionally, there is employed a method in which a sample containing an analyte is properly diluted and subjected to a qualitative reaction with a measuring reagent having a predetermined sensitivity and a maximum dilution ratio at which positivity is shown is multiplied by the sensitivity to determine a semi-quantitative assay value, or qualitative reactions are carried out with reagents having different sensitivity without diluting an analyte and the sensitivity of the reagent at which the analyte shows positivity is taken as a semi-quantitative assay value. The method of quantitative analysis includes a liquid-phase assay carried out in a container such as a microtiter well and a solid-phase assay carried out on a chromatographic medium. In the methods described in the above two publications, samples are diluted for carrying out the quantitative determination.
JP-A-4-351962, laid-open recently, discloses a specific-binding analysis method which permits semi-quantitative assay without diluting a sample and an apparatus therefor. In the method described in this publication, for the qualitative or quantitative determination of an analyte in a sample by a chromatographic method, a specific substance is allowed to be present in a measurement system and the amount of a labeled substance to be measured as an index for the analyte is decreased due to the presence of the specific substance, so that there can be consequently obtained a result similar to the result obtained by diluting a sample containing the analyte (to be referred to as xe2x80x9cdilution effectxe2x80x9d hereinafter).
In a typical method described in the above publication, an analyte (a) added to a sample addition portion contacts a predetermined amount (concentration) of a specific substance (b), which is present on a chromatographic material without being fixed, and a predetermined amount of a labeled specific-binding substance (e), which is present on the chromatographic material without being fixed, (in a site where the specific-binding substance is present). A certain amount of the analyte (a) bonds to the specific substance (b). When, however, an excess of the analyte (a) over the specific substance (b) is present, the excess of the analyte (a) moves to a portion (detection portion) where a specific-binding substance (specific substance (b) or a substance (g) capable of binding to that portion of the analyte (a) which the specific substance (b) bonds to) is present being fixed to the chromatographic material while the excess of the analyte (a) retains a portion capable of binding to the specific substance (b). The analyte (a) which has no portion capable of binding to the specific substance (b), or at least has bound to the specific substance (b), passes the detection portion. Only a complex (f) of the analyte (a)xe2x80x94the labeled specific-binding substance (e), which at least has a portion capable of binding to the specific substance (b), is fixed on the detection portion, and this fixed complex (f) is detected by various means.
It has been generally required to dilute a sample for semi-quantitative assaying an analyte in the sample. Further, the method using reagents having different sensitivity, which does not require the dilution of a sample, has a defect in that the determination is difficult since the intensity of one reaction in one measurement sensitivity is different from the intensity of another reaction in another measurement sensitivity, so that this method has been scarcely practically used.
In the field of medical treatment, clinical examination in particular, the operation of diluting a sample increases the possibility of an examiner being infected with pathogen from blood, urine, etc., as testing samples. Further, when a large number of samples are semi-quantitative assayed, the operation efficiency can be remarkably improved if the dilution operation is not necessary. It is therefore an object of the present invention to provide a simple immunochemical semi-quantitative assay method which does not require the dilution of a sample, and an apparatus therefor.
Like the present invention, the above-described JP-A-4-351962 discloses an invention which does not require the dilution of a sample. In the method of this publication, many factors are concerned with the adjustment of xe2x80x9cdilution effectxe2x80x9d of a sample, and it is difficult to adjust the dilution effect, so that no narrow semi-quantitative assay value width can be set and that the method has a problem in measurement accuracy. It is further an object of the present invention to provide an immunochemical semi-quantitative assay method in which the number of factors concerned with the adjustment of xe2x80x9cdilution effectxe2x80x9d is small so that the dilution effect can be easily adjusted and the sensitivity can be improved, i.e., a semi-quantitative assay value width can be narrowly set, and an apparatus therefor.
For achieving the above objects, the present inventor has made diligent studies, and has found that the above objects are achieved by a simple immunochemical semi-quantitative assay method according to chromatography, which comprises trapping a certain amount of an analyte in a sample with a predetermined amount of a fixed antibody for the analyte before qualitative analysis of the analyte, the certain amount corresponding to the amount of the fixed antibody, and thereby decreasing a concentration of the analyte to be subjected to subsequent immunochemical qualitative determination, and a simple immunochemical semi-quantitative assay apparatus according to immunochromatography, which comprises a sample addition portion (A) for adding a sample containing an analyte, an antibody-fixed portion (B) where an antibody for trapping a certain amount of the analyte in the sample is present being fixed in a predetermined amount, a labeled substance presence portion (C) where a labeled substance as an index for detecting the presence or absence of the analyte in the sample is present in a chromatographically mobile state, a detection portion (D) where a detecting substance for detecting the labeled substance is present being fixed, and an absorption portion (E) for removing the labeled substance which does not participate in the detection of the analyte, together with the added sample, by absorption.
In the semi-quantitative assay method and the apparatus of the present invention, in a series of chromatographic analysis, a certain amount of an analyte in a sample is trapped with a fixed antibody before qualitative determination so that the amount (concentration) of the analyte in the sample to be subjected to qualitative determination is decreased in advance, whereby the same effect (dilution effect) as that produced by qualitative determination by immunochromatography after the dilution of the sample containing the analyte can be obtained.
That is, the assay is carried out by adding the same sample to several units in which the fixed amounts of a trapping antibody are changed stepwise (the sample being to have a different dilution degree in each unit), whereby, in the subsequent qualitative analysis in each unit having common sensitivity, the semi-quantitative assay value of an analyte in the sample can be determined on the basis of the fixed amount of the trapping antibody set at the unit in which the analysis result changes from positivity to negativity or from negativity to positivity.